Bacteraemia and obstructive pyelonephritis caused by Bifidobacterium breve in an elderly woman: a case report and literature review

Bifidobacterium spp. are non-spore-forming Gram-positive anaerobes that are indigenous to the human gastrointestinal tract and vagina. They are believed to be non-pathogenic organisms for humans and thus are widely used as probiotics. An 83-year-old woman taking cephalexin for 4 days was diagnosed with obstructive pyelonephritis. Y-branched Gram-positive rods were found in both anaerobic and aerobic blood culture bottles, and in an anaerobic urine culture. Bifidobacterium breve was finally identified. Ceftriaxone and metronidazole were administered to the patient, and she was discharged after intermittent catheterization for dysuria. Urinary tract infection caused by Bifidobacterium spp. is believed to be rare, but it can develop in patients with underlying urological conditions. Recognition of the characteristic morphology and conducting anaerobic urine culture may help in identifying more cases of Bifidobacterium urinary tract infections.


INTRODUCTION
Bifidobacterium spp.are non-spore-forming Gram-positive anaerobes that are indigenous to the human gastrointestinal tract and vagina.There are over 50 Bifidobacterium species; however, only 11 have been isolated from the human gut and oral cavity [1].
The patient was diagnosed with obstructive pyelonephritis and admitted to the hospital after foley catheter insertion.Therefore, ceftriaxone 1 g qd was administered intravenously to the patient after obtaining samples for a urinary culture and two sets of blood cultures.The following day, a urologist was consulted.Based on imaging findings and bacteriuria, the urologist determined that she had chronic cystitis.The cause of the obstructive pyelonephritis was unclear; however, invasive procedures were not indicated, considering the patient's general condition.
Two sets of anaerobic blood culture bottles (BD BACTEC 22F anaerobic medium; Becton Dickinson and Company, Sparks, NV, USA) and one set of aerobic culture bottles (BD BACTEC 23F aerobic medium) gave positive results after 33, 47 and 132 h, respectively.Gram staining from the blood culture bottles revealed Gram-positive rods (GPRs) with Y-branched forms (Fig. 1).The GPRs were incubated at 35 °C in an anaerobic atmosphere on an ABHK agar plate (Nissui Pharmaceuticals, Tokyo, Japan) and in an aerobic atmosphere supplemented with 5 % CO 2 using BBL trypticase soy agar (TSA) with 5 % sheep blood and chocolate II agar LDIP (Becton Dickinson and Company, Sparks, NV, USA).Grey and smooth colonies were observed on the ABHK agar plates after 48 h (Fig. 2).Colonies smaller than those on ABHK agar were found on BBL TSA agar after a 48 h incubation period.All bacteria developed on ABHK agar plates and TSA with 5 % sheep blood and chocolate II agar were identified by two matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) methods.They were identified as B. breve using MBT Compass v. 4.1 and MBT Compass Library v. 9.0.0.0.(8468MSPs) (Bruker Daltonics, Bremen, Germany; score value 2.11) with a microflex LT/ST system, and as Bifidobacterium spp.using VITEK MS software 4.3.0 and VITEK MS Knowledge Base version 3.0 (bioMérieux, Marcy-l'´Etoile, France).The 16s RNA sequences of the amplified products obtained from the organism matched 100 %.
Following catheter drainage and administration of ceftriaxone, the patient soon became afebrile.The clinical course of this case proceeded well.After the organism was identified as B. breve, oral metronidazole 500 mg TID was added to the treatment.Ceftriaxone and metronidazole were administered for 11 and 4 days, respectively.An interview with the patient revealed no history of probiotic or dairy product use.On day 29, the patient was discharged after intermittent catheterization for dysuria.
Two days after discharge from the hospital, the patient was readmitted because of a recurrence of obstructive pyelonephritis, and only extended-spectrum β-lactamaseproducing K. pneumoniae (MIC: ceftriaxone >1 µg ml −1 , meropenem≦0.25 µg ml −1 , levofloxacin 1 µg ml −1 ) were isolated from blood and urine cultures.During the hospital stay, she developed cerebral embolism and died approximately 7 weeks after the B. breve bacteraemia episode.

DISCUSSION
The clinical significance and incidence of infections caused by Bifidobacterium spp.are unclear.In Norway, 0-2 bacteraemia cases due to Bifidobacterium spp.were reported annually between 2007 and 2012 [6].Brook et. al. reported that 57 Bifidobacterium spp.were identified in 2033 samples from paediatric patients.Among these, Bifidobacterium spp.could not be identified in 342 blood cultures [8].Mahlen et al. reported three adult cases of B. breve bacteraemia during 2000-2007 in two US hospitals [10].Boume et al. reported that 10 Bifidobacterium spp.were detected in 91 493 blood cultures from 1972 to 1977 [12].Probiotics play a major role in Bifidobacterium bloodstream infections in children [13,14].However, it is unclear whether invasive Bifidobacterium infections in adults are associated with probiotics [15].
In total, 14 cases of bloodstream infections with B. breve have been reported in the literature (Table 1) [3,6,10,13,16,17].Although Bifidobacterium spp.are classified as anaerobes, they can grow in aerobic atmospheres supplemented with 5 % CO 2 .Andriantsoanirina et al. reported that many Bifidobacterium spp.are resistant to oxygen; in fact, 77.8 % of the evaluated B. breve strains were resistant to oxygen [18].Here, the time to positivity from the blood culture was longer for the aerobic bottles than that for the anaerobic bottles.Moreover, B. breve bacteraemia might be underestimated in children taking probiotics because usually only samples for aerobic cultures are collected from children.To our knowledge, UTIs caused by Bifidobacterium spp.are quite rare; we only found nine cases (Table 2) [5,10,[19][20][21][22][23].
Most of the patients had underlying diseases and urological problems.We suspected the following aetiology for the present case: chronic cystitis led to obstructive pyelonephritis caused by polymicrobial pathogens; preceding cephalexin selected B. breve, which is naturally resistant to cephalosporins; and, B. breve bacteraemia developed due to increased pressure in the renal pelvis.Here, Gram staining of urine specimen showed numerous typical Y-branched GPRs; however, no growth was observed in the routine urine culture.Anaerobic urine culture should be considered in patients with an immunocompromised state, urological problems and positive Gram staining but negative routine culture [24].If Bifidobacterium spp.are suspected based on Gram staining and poor growth of routine urine culture, anaerobic culture is the key to the early identification of this organism.
Sequencing of the 16s RNA gene has been used as the gold standard for accurate species identification [1,25].However, genetic analysis is difficult to perform in actual clinical practice.The identification of Bifidobacterium spp.based on phenotypic characteristics is challenging [26].Identification by biochemical testing is known to be affected by insufficient growth and poor reproducibility [1].MALDI-TOF-MS has been reported to be useful in identifying anaerobic bacteria, including Bifidobacterium spp.[29,30].
The performance of MALDI-TOF-MS has been believed to surpass that of these commercial kits, with a higher rate of correct identification and fewer misidentifications.As such, the implementation of MALDI-TOF-MS has become a cornerstone in the identification of anaerobic bacteria, including our hospital [1,31].

CONCLUSION
In this report, we describe a rare case of B. breve bacteraemia and obstructive pyelonephritis.The combination of Bifidobacterium bacteraemia and UTI is believed to be rare; however, there may be undiagnosed cases due to the poor growth in routine urine culture and the difficulty of identification.Recognition of Y-branched GPR and conducting anaerobic urine culture may lead to find more cases of Bifidobacterium UTIs.Clinicians and microbiology technicians need to keep in mind the usefulness and limitation of commercial kits and MALDI-TOF-MS in identifying Bifidobacterium spp.
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Comments: 1. Description of the case(s): Case has been described in sequential manner with major and relevant findings.However, there are a few points as detailed below: in Case presentation Page 3 L16-20 opening statememt of case presentation can be rephrased eg."An 83-year-old woman with diabetes mellitus and dementia presented in our hospital with Fever, loss of appetite, inability to move and impaired consciousness.As per available information, four days earlier, she had visited a local clinic with complaints of fever and was prescribed with cephalexin.She gradually developed loss of appetite and difficulty in ambulation.Fever & loss of appetite persisted even after four days and she could not move.She was then shifted to the hospital.Other details could not be found as the patient was having dementia and impaired consciousness."L23-24 "Neither abdominal tenderness nor costovertebral tenderness was noted in the patient " This sentence should be rephrased as "The patient did not have abdominal tenderness and costovertebral tenderness."To convey the message correctly.Page 3, L30 "however, neither ureteral nor kidney stones" to be rephrased as "with no kidney or ureter stones.L32-delete "therefore" page 4 L16-"Gram staining of the urine samples showed only numerous Y-branched GPRs" to be written as "Gram staining of the urine sample showed numerous Y-branched GPRs." (delete only and Urine sample instead of urine samples) Conclusion: page 6 L13-"Recognition of Y-branched GPR" to be written as Recognition of Y-branched GPR in Gram stain Page 6 L14-conducting anaerobic urine culture may lead to find more cases of Bifidobacterium UTIs ----conducting anaerobis urine culture may lead to finding more cases of Bifidobacterium UTIs. 2. Presentation of results The results are now presented in sequential manner as per the order of events 3. How the style and organization of the paper communicates and represents key findings The organization and style is now sequential as per order of events with relevant significant clinical details, investigations, course of event during hospital stay etc. 4. Literature analysis or discussion literature has now been analysed and discussed well 5. Any other relevant comments The Case report

Please rate the quality of the presentation and structure of the manuscript Good
To what extent are the conclusions supported by the data?Strongly support

Introduction
I think this should be longer, and some of the points you make should be elaborated.For example, in line 4 and line 6 it would be nice to know if these 10 species were infection or have been identified as part of microbiome studies, and what other infections have been reported.Can you please include further references.

Response:
We thank the reviewers for their suggestions.We changed the Introduction.We re-checked reference 6 (J Clin Microbiol.2017;55 (7):2234-48); we could not find the original source of the description.Manual of Clinical Microbiology Ed.11 & 12 also described similar content; however, we could not track back to the original article due to the lack of a reference.Thus, unfortunately, we cannot describe the details of the 10 species per your comment.

Case Presentation
Line 11 -can you explain why she was prescribed cephalexin?What was the basis of this choice of empirical antibiotic?Were there any clinical signs of UTI?

Response:
We did not receive a referral note for this case.We guess the previous doctor suspected a urinary tract infection (UTI), because cefalexin is often prescribed to treat UTIs in our country.Because we do not know the doctor's name and the details, we think we should not write the uncertain reason for the prescription in the body of the text.The descriptions of back pain and CVA tenderness were not found in the medical record.Please see Page 3, Line 20 & 27-28.
Lines 14-21 -as this is a Microbiology journal, and the audience will likely include non-clinicians, can you please state the relevance of these results as they appear (ie.Are they high/low/abnormal).

Response:
As you suggested, we added interpretations of physical examination and laboratory data.Please see Page 3, Line 25-33.
Line 26 -"The patient's clinical course after admission was uneventful." this needs to be reworded, as a full clinical outcome is noted on page 4 line 16 onwards.

Response:
Reviewer 2 also commented on the same point, thus we deleted it.
Line 27 -you state she had 2 sets of blood cultures but you do not mention whether these were collected before or after the ceftriaxone -this is important and needs to be included.

Response:
We added the details of the culture collection.Page 4 Line 1-3.
Line 36 -smaller colonies found on the BBL TSA -I presume these were also identified as the same organism?Can this be added if so.

Response:
As you suggested, we added the description of identifications as follows: "All bacteria developed on ABHK agar plates and TSA with 5% sheep blood and chocolate II agar were identified by two matrix-assisted laser desorption/ionization time-of-flight mass spectrometry methods (MALDI-TOF-MS).

Response:
We changed the description of the 16s RNA sequence result as follows: "The 16s RNA sequences of the amplified products obtained from the organism matched 100%." Page 4, Lines 24-25

Discussion
Overall, this is a decent discussion but it can be strengthened a great deal.The literature has obviously been reviewed by the authors, but the data hasn't been presented as well as it could in the text -it would benefit from some summarisation.
Line 28 -"0-2 cases" -reword this; is it 2 cases from 2007-2012, is it 2 cases every year, or can you work out the total number of cases over that time period.Is Norway the only country to report cases?

Response:
According to Reference 6 (Esaissen et al), 0-2 Bifidobacteriumbacteremia cases were reported annually in Norway.The article did not have a reference for the number.We could not find the incidence of Bifidobacteriumbacteremia in other countries.Alternatively, we cited two references on the epidemiology of Bifidobacteriumbacteremia. Please see Page 5, Line 16-21.
Table 1 and table 2 could easily be combined so that you are reviewing all reported cases, then refer to UTI separately and reference the articles.

Response:
We have reviewed your proposal carefully.However, we believed Tables 1 and 2 should be separate for the following reasons.First, Table 1 deals with B. brevebacteremia.B. brevewas isolated from aerobic bottles and was often reported in infants taking probiotics.
We hope to include information on blood cultures (system, bottle, and time to positivity).Second, Bifidobacteriumurinary tract infections are thought to be rare, but there are many cases with underlying urinary tract diseases and a history of UTI.To make the clinical picture more easily understandable, we hope to keep the original Tables.
Line 7 page 5 -please clarify that this is a hypothesis for *this case* -make that clear.Did the patient say in the initial work up that she had been experiencing chronic cystitis, as you haven't mentioned this previously.

Response:
As you indicated, we emphasized that this is a hypothesis about "the present case".Page 5, Line 36.
Actually, we did not obtain a history of chronic cystitis.We added the following sentence: "Based on imaging findings and bacteriuria, the urologist determined that she had chronic cystitis." Page 4, Line 3-4 Line 11-12 page 5 -was the urine sample taken at the same time as the blood cultures?Was this pre-ceftriaxone?Add "urine" to routine culture and state GPR were seen in the urine specimen Gram stain.

Response:
Urine and blood cultures were collected before the administration of ceftriaxone.Please see Page 4, Line 1-3 We added "urine" to "routine culture", and "of urine specimen" to "Gram staining showed".Page 6, Line 5-8.
Line 15 page 5 -"Few reports" how many is few?You've reviewed the literature so this number should be known?

Response:
We have stated this in the sense that there have been few studies of only Bifidobacteriumspp. in clinical specimens.However, since this could be misleading, we have added the position of the 16sRNA sequence and the description of MALDI-TOF-MS in the identification of Bifidobacteriumspp.Page5, Line 10-.
Line 23 page 5 -I think this is a really important point that you should definitely highlight with more gravitas.Diagnostic labs without a MALDI-ToF may struggle to identify these isolates if relying on Vitek, as API tests are becoming less common (from my experience in the UK).

Response:
As suggested, we noted the following sentences: "If microbiology technicians think that anaerobic bacteria are absent due to their growth in aerobic bottles, an incorrect identification kit may be used, which can result in misidentification of the bacteria.
Although identifying the bacteria to species level by biochemical characterization is sometimes difficult, identification to the genus level is possible if the correct identification kit is selected.Characteristic Gram staining will help in identifying the Bifidobacteriumspp.without MALDI-TOF-MS or sequencing of the 16s RNA gene."

Conclusion
The conclusion needs to be reworded, or the results section needs to clearly include that the two commercial kits mentioned in the discussion were also tested, as this information isn't stated.This is the final statement of the case report: "A greater compilation of Bifidobacterium infection reports is necessary to clarify the pathogenicity, clinical picture, and optimal management of infections, especially UTI."The final statement of your conclusion should be the importance of the case you have reported and the important points you have discovered and you think are important for clinicians and scientists to know.If you want to keep this statement it might be worth having this in the discussion instead.

Response:
Thank you for your suggestion.We revised the conclusion as follows."In this report, we describe a rare case of B. brevebacteremia and obstructive pyelonephritis.The combination of Bifidobacteriumbacteremia and UTI is believed to be rare; however, there may be undiagnosed cases due to the poor growth in routine urine culture and the difficulty of identification.Recognition of Y-branched GPR and conducting anaerobic urine culture may lead to finding more cases of BifidobacteriumUTIs. Clinicians and microbiology technicians need to keep in mind the usefulness and limitations of commercial kits and MALDI-TOF-MSin identifying Bifidobacteriumspp." We also move the sentence "A greater compilation of …. ".Page 7, Line 7-8

Response to the comments of Reviewer #2
We wish to express our sincere appreciation to the reviewer for the insightful comments that have helped us significantly improve the quality of our paper.We have addressed the comments with point-by-point responses and revised the manuscript accordingly.Unfortunately, the patient died, so we could not obtain more information from them.
During revision, we found two B. brevebacteremia and one new case of BifidobacteriumUTI, thus we changed the total number of UTI cases and Table 2.
Please rate the quality of the presentation and structure of the manuscript Abstract to be rewritten-with brief introduction of Bifidobacterium spp , importance/ role in causing disease and then brief introduction of case being reported and its significance/importance.

Response:
Thank you for your suggestion.We revised the abstract.Page 2.
Page 1, L12-13: Presenting complaints in hospital to be written instead of "she presented to our hospital in an ambulance." Relevant clinical history & Physical examination findings with respect to urological complaints or disorders/pre-existing urinary tract structural abnormalities or obstructions etc. to be mentioned.
Other relevant investigations and findings (even negative ones) to be mentioned eg.RBCs, casts & crystals in urine, X ray KUB, USG abdomen & pelvis,

Response:
As suggested, we noted her status before admission.Page 3, Line 21-23.Actually, we did not obtain any urological complaints due to dementia and impaired consciousness.We added the descriptions of abdominal tenderness/CVA tenderness, urinalysis, and radiologic findings.Page 3, Line 27-28.
Page 1, L23: therefore to be deleted, Dose & route of administration of Ceftriaxone to be mentioned.
Timing of collection of blood cultures and urine culture to be mentioned as per sequence of events

Response:
We added information on ceftriaxone and culture collections."Therefore, ceftriaxone 1 g qd was administered intravenously to the patient after obtaining samples for a urinary culture and 2 sets of blood cultures." Page 4, Line 1-3 Page 1 L26: "The patient's clinical course after admission was uneventful" clinical course in hospital, follow up investigations if any, duration of hospital stay & outcome etc to be mentioned in the case description.

Response:
Reviewer#1 also mentioned "The patient's clinical course…", thus we deleted the sentence.However, we noted that "she became afebrile soon." Actually, we did not have follow-up investigations during the clinical course.We added the outcome of this case at the end of the case.She developed pyelonephritis due to ESBL-producing K. pneumoniae, but this was another episode.Page 4, Line 8-12.
31gram staining from blood culture bottles

Response:
As suggested, we added "from blood culture bottles" to the sentence.Page 4, Line 10.
Page 2, L-2: "The organism was identified as B. breve " -should be written as The organism was identified as Bifidobacterium breve.

Response:
We already noted "Bifidobacterium breve"in the Introduction.We think the abbreviated form "B. breve" is better since it is more concise in this sentence.We have defined B. breveas a short form of Bifidobacterium breveat first mention on page 3 lines 12 Page 2 L-11 to L-15: regarding urine sample, there is no mention of urine sample subjected to culture (aerobic &/or anaerobic), their numbers, timing etc. Antimicrobial sensitivity of aerobic and anaerobic isolates from urine to be mentioned.

Response:
As suggested, we noted the details and essential MIC of Bifidobacterium breveand other bacteria.Antimicrobial susceptibility testings of Enterococcus faecalisand Murdociellasp.were not performed.These should be linked and described together.

Response:
Reviewer#1 also mentioned two MALDI-TOF-MS results.We changed the position of the description of the two MALDI-TOF-MS results.Please see Page 3, Line 20-25.
Page 2, L17-18: dosage of ceftriaxone and metronidazole also to be mentioned.

Response:
We added a description of the dosage of metronidazole.We mentioned the dosage of ceftriaxone in the previous sentence.
The organization and style to be sequential as per order of events with relevant significant clinical details, investigations, course of disease/event during hospital stay and outcome etc. as detailed above We changed the structure of the paper.As for case presentation, we noted the case description -microbiological testsubsequent case description.

Response:
Thank you for your comment.Per your comment, we changed the case description, microbiological test, and subsequent case description.

Literature analysis or discussion
Page 4, L-33: " bloodstream infections with B. Breve" should be written as bloodstream infections with Bifidobacterium breve

Response:
We think "bloodstream infections with B. breve" is better since it is more concise.We have defined B. breveas a short form of Bifidobacterium breveat first mention on page 2 lines 12-13.
Page 5, L17-L23: two commercial kits have been mentioned for identification of Bifidibacterium spp.However, their use or reason for not using in the present case is not discussed.

Response:
In this case, we initially used Microflex® LT/ST and could identify B. breveat first.When we decided to write the case report, we needed to check the identification performance of other MALDI-TOF-MS (VITEK-MS) and 16s RNA sequence.
Actually, we seldom use these commercial kits to use to identify the anaerobes.Thus, we had only expired API®RAPID ID 32A when we receive the reviewer's comment.We used API®RAPID ID 32A using preserved B. breve.The results were as follows: Actinomyces naeslundii69.2%,Propionibacterium propionicum26.7%.
We are not sure the reason of misidentification, however, expired kits, preserved bacteria, and irregular phenotype may lead to the result.We confirmed the identification of the bacteria by 16s RNA sequencing.The result may confuse readers, thus we do not describe the result.We added the sentences of limitation of commercial kits.Page 6, Line 13-14 Page 5, L24-L27: in discussion, experience in identification to species level in this case with MALDI-TOF (Microflex LT instrument, Bruker Daltonics) is not mentioned although the same is written in case description.
" identification of B. breve was finally re-confirmed by 16s 24 rRNA sequencing" is not discussed with respect to literature.

Response:
As suggested, we revised the discussion section about identification by 16s RNA and MALDI-TOF-MS.Please see Page 6, Line 30-

Any other relevant comments
Anaerobic culture are routinely not done in cases of pyelonephritis.possibility of anaerobic pathogen to be kept in mind.Anaerobic cultures to be done in patients with high index of suspicion especially in elderly patients with immunocompromised state or Comments: [Delete this text before submitting your review.Please include comments to the author here, and include the below sections, where possible.All comments here will be posted publicly online alongside the article once the Editor has made a decision.]1. Description of the case(s) Abstract to be rewritten-with brief introduction of Bifidobacterium spp , importance/ role in causing disease and then brief introduction of case being reported and its significance/importance. Page 1, L12-13: Presenting complaints in hospital to be written instead of "she presented to our hospital in an ambulance."Relevant clinical history & Physical examination findings with respect to urological complaints or disorders/pre-existing urinary tract structural abnormalities or obstructions etc. to be mentioned.Other relevant investigations and findings (even negative ones) to be mentioned eg. ).These should be linked and described together.Page 2, L17-18: dosage of ceftriaxone and metronidazole also to be mentioned.3. How the style and organization of the paper communicates and represents key findings The organization and style to be sequential as per order of events with relevant significant clinical details, investigations, course of disease/event during hospital stay and outcome etc. as detailed above 4. Literature analysis or discussion Page 4, L-33: " bloodstream infections with B. Breve" should be written as bloodstream infections with Bifidobacterium breve Page 5, L17-L23: two commercial kits have been mentioned for identification of Bifidibacterium spp.However, their use or reason for not using in the present case is not discussed.Page 5, L24-L27: in discussion, experience in identification to species level in this case with MALDI-TOF (Microflex LT instrument, Bruker Daltonics) is not mentioned although the same is written in case description." identification of B. breve was finally re-confirmed by 16s 24 rRNA sequencing" is not discussed with respect to literature. 5. Any other relevant comments Anaerobic culture are routinely not done in cases of pyelonephritis.possibility of anaerobic pathogen to be kept in mind.Anaerobic cultures to be done in patients with high index of suspicion especially in elderly patients with immunocompromised state or debilitating illness or underlying urological conditions for iidentification of anaerobic infections and appropriate treatment to improvei patient outcome.comment to this effect to be included in conclusion.

Please rate the quality of the presentation and structure of the manuscript Satisfactory
To Comments: Thank you for the opportunity to review this case report that is presenting a rare case of Bifibobacterium breve from blood cultures with a urinary origin.This is an interesting case and you have reported the clinical picture, laboratory diagnosis, and management of the patient.The language used overall is good, but in some places is too abrupt and does not elaborate on finer details. References in text need to be with the statement and not at the beginning of the following sentence.The key findings of the case report need to be highlighted in the conclusion.Introduction I think this should be longer, and some of the points you make should be elaborated.For example, in line 4 and line 6 it would be nice to know if these 10 species were infection or have been identified as part of microbiome studies, and what other infections have been reported.Can you please include further references.Case Presentation Line 11 -can you explain why she was prescribed cephalexin?What was the basis of this choice of empirical antibiotic?Were there any clinical signs of UTI? Lines 14-21 -as this is a Microbiology journal, and the audience will likely include non-clinicians, can you please state the relevance of these results as they appear (ie.Are they high/low/abnormal).Line 26 -"The patient's clinical course after admission was uneventful." this needs to be reworded, as a full clinical outcome is noted on page 4 line 16 onwards.Line 27 -you state she had 2 sets of blood cultures but you do not mention whether these were collected before or after the ceftriaxone -this is important and needs to be included.Line 36 -smaller colonies found on the BBL TSA -I presume these were also identified as the same organism?Can this be added if so.Lines 21-24 on page 4 -can you please put the 16S results after the initial MALDI-ToF result, and can you please state where the 16S sequencing was performed.
Can you please write something to the effect of "100% sequence match" instead of just 100%.Discussion Overall, this is a decent discussion but it can be strengthened a great deal.The literature has obviously been reviewed by the authors, but the data hasn't

Reviewer 2 :Reviewer 2
SatisfactoryTo what extent are the conclusions supported by the data?Reviewer 2: Strongly support Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?Reviewer 2: No: If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?Reviewer 2: Yes: Comments to Author: 1. Description of the case(s)
RBCs, casts & crystals in urine, X ray KUB, USG abdomen & pelvis, Page 1, L23: therefore to be deleted, Dose & route of administration of Ceftriaxone to be mentioned.Timing of collection of blood cultures and urine culture to be mentioned as per sequence of events Page 1 L26: "The patient's clinical course after admission was uneventful" clinical course in hospital, follow up investigations if any, duration of hospital stay & outcome etc to be mentioned in the case description.L30-31, gram staining from blood culture bottles 2. Presentation of results: should be seuential.Page 2, L-2: "The organism was identified as B. breve " -should be written as The organism was identified as Bifidobacterium breve.Page 2 L-11 to L-15: regarding urine sample, there is no mention of urine sample subjected to culture (aerobic &/or anaerobic), their numbers, timing etc. Antimicrobial sensitivity of aerobic and anaerobic isolates from urine to be mentioned.For Page2, L2-L5: "The organism was identified as B. breve based on matrix-assisted laser 3 desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS, microflex ® 4 LT/ST using MBT Compass Ver 4.1 and MBT Compass Library Ver 9.0.0.0.(8468 5 MSPs) (Bruker Daltonics, Bremen, Germany; score value 2.11)." and L21-24: "The bacterium was identified as Bifidobacterium spp.by MALDI-TOF-MS (VITEK MS 22 using the software 4.3.0 and VITEK MS Knowledge Base version 3.0, bioMérieux, Marcy.23 Marcy-l'´Etoile, France

Table 1 .
Reviews of bacteremia caused by
According to the manufacturer's instructions, the three commercial kits that can identify Bifidobacterium spp. in each database based on biochemical characterization are the BD BBLTM CRYSTALTM ANR ID System (Becton Dickinson and Company, Sparks, NV, USA), which can identify B. adolescentis, B. dentium and Bifidobacterium spp.; the API 20A (bioMérieux, Marcy-l'Etoile, France), which can identify B. adolescentis, B. dentium, B. breve, B. bifidum and Bifidobacterium spp.; and the API RAPID ID 32A (bioMérieux, Marcy-l'Etoile, France), which can identify B.
adol28]entis, B. breve, B. longum, B. dentium, B. bifidum and Bifidobacterium spp.[27,28].If microbiology technicians think that anaerobic bacteria are absent due to their growth in aerobic bottles, an incorrect identification kit may be used, which can result in misidentification of the bacteria.Although identifying the bacteria to species level by biochemical characterization is sometimes difficult, identification to the genus level is possible if the correct identification kit is selected.Characteristic Gram staining will help in identifying the Bifidobacterium spp.without MALDI-TOF-MS or sequencing of the 16s RNA gene.
They were identified as B. breveusing MBT Compass Ver 4.1 and MBT Compass Library Ver 9.0.0.0.(8468MSPs) (Bruker Daltonics, Bremen, Germany; score value 2.11) with the Microflex ® LT/ST system, and as Bifidobacteriumspp.usingVITEK MS software 4.3.0 and VITEK MS Knowledge Base version 3.0 (bioMérieux, Marcy-l'´Etoile, France).The 16s RNA sequences of the amplified products obtained from the organism matched 100%."Page4, Lines 17-25Lines 21-24 on page 4 -can you please put the 16S results after the initial MALDI-ToF result, and can you please state where the 16S sequencing was performed.Can you please write something to the effect of "100% sequence match" instead of just 100%.
what extent are the conclusions supported by the data?Strongly supportDo you have any concerns of possible image manipulation, plagiarism or any other unethical practices?NoIs there a potential financial or other conflict of interest between yourself and the author(s)?NoIf this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?YesReviewer 1 recommendation and comments https://doi.org/10.1099/acmi.0.000574.v1.4 © 2023 Cleaver L. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.King's College London, Centre for Host Microbiome Interactions, Floor 17, Tower Wing, Guy's Hospital, Great Maze Pond, London, UNITED KINGDOM https://orcid.org/0000-0002-7788-9384